Atropa belladonna

A very toxic plant grown as a source of the tropane alkaloids scopolamine, atropine, and hyoscyamine.

Propagation

Germination

Positive photoblasty.[1]

In a 1967 study, germination in belladonna seeds scarified with concentrated sulfuric acid for 1 minute was said to have occurred in three weeks (no data).[3] An earlier paper by the same research group using the same protocol suggested the period was “two to three weeks” for germination.[4] This is, in contrast, a 2012 later study that showed no germinations with their 1-minute sulfuric acid treatment, though the concentration was not given (60% in the 2-minute treatment).[2]

Mechanical scarification by dissection and removal of the seed coat increases germination rates, but it is not practical due to the small size of the seeds and the high risk of damage.[2]

In a diverse study of seed pretreatments, boiling water, nitric acid, GA3 were reportedly successful at increasing the speed and rate of germination. The authors recommend nitric acid treatment for 3 minutes, though the concentration of the acid was not given. Boiling water treatment (20 min) resulted in germination after two days with a final rate of 90% on MS media. However, only ten seeds were used¹ per treatment casting doubt on the statistical significance of these results.[2]

[5]

Vegetative

Belladonna will self-propagate vegetatively.[6]

In-Vitro

[2] [7]

Cultivation

With acid scarification, belladonna plants can achieve a height of 2-3 cm with five leaves after approximately 6 weeks (3 weeks germination). This was sufficient to transfer these seedlings to hydroponic culture.[3]

Harvest

Harvest should be timed to avoid wet conditions. Plants should be dried quickly to avoid degradation of the alkaloids.[9]

Belladonna can be cut 15-20 cm above the ground for harvest leaving several leaves intact. The whole shoot can be hung to dry.[8]

Yield

Plants are typically cultivated for two or three years. Leaves are harvested at the end of the first year. At the end of the second or third year both leaves and roots are fully harvested for a significantly higher yield.[9]

Seeds collected from the previous year’s largest fruit fraction yield more than those from the smallest fruit fraction.[6]

Flowering begins later in first-year crops leading to a significantly smaller yield of seeds compared to second and third-year crops.[6]

Belladonna growing in sunny locations gives a higher yield.[6]

Severe dehydration stress can moderately increase the concentration of alkaloids in belladonna with complex interactions of nitrogen supply. This comes at a more than four times reduction in biomass, however.[10]

Soilless

[3]

Soil

Fertilization

In a study of water stress and nitrogen fertilization, watering at 35% depletion of available soil water (reduction from field capacity) yielded the highest root mass with dose-dependent diminishing growth with further dehydration.[10]

While no single chemical fertilizer is sufficient to increase the concentration of alkaloids in belladonna, the combination of high nitrogen and high phosphorus can. Nitrogen itself can increase the biomass yield of belladonna without diminishing alkaloid concentration.[8] However, later research reports an interaction between the ratio of calcium and potassium alone, leading to possible confounding due to the use of calcium phosphate and calcium-based lime in multiple trials.[3] The increased production of alkaloids in the high nitrogen and “high phosphorus” trial could be the result of high calcium instead. Regardless, the bone meal might be a suitable organic fertilizer specially tuned for belladonna production. Though it is important to note that the difference between the various trials was minimal, but significant nonetheless.[3]

[8]

Temperature

[11]

Lighting

Pests

Ecology

Morphology

Roots

Stem

Leaves

Inflorescence

Seeds

Phytochemistry

[7]

Infraspecific Variation

Biosynthesis

[12] [13]

Distribution

Tropane alkaloids are likely synthesized in the roots and transported to the leaves. [14][15] However, unlike alkaloids in Nicotiana spp., hyoscyamine and scopolamine are not respondents to wound-induced upregulation or jasmonic acid treatment.[16]

The relative distribution of alkaloids varies with time of day, plant age, and time of year.[12]

[12] [13] [7]

Timecourse

Tropane alkaloids in belladonna have a diurnal variation of roughly 300%. This effect could be a byproduct of the continuous degradation of hyoscyamine to the less active racemate, atropine, and the need for the plant to continuously recycle the tropane skeleton to produce more active L-hyoscyamine de novo. The variation stems from relative synthesis and transport rates between the roots and shoots during transpiration.[12]

[12] [13]

Improvement

Identification

Inheritance

Methods

History & Society

Work Log

11 Jun 2023

04 Jun 2023

09 Feb 2023

Placed 15 SM seeds into a 50ml falcon tube with 25 ml of water. Put into refrigerator for artificial cold stratification. Set a timer for 4 weeks.

Attempting acid scarification of the remaining 16 seeds based on the procedure of Asha and Prasad 2012. The original protocol used nitric acid for 3 minutes giving 95% germination in a matter of days. However, the acid concentration was not given. If they used dilute acids I run the risk of destroying my seeds using the same protocol with more concentrated acids. I would like to try the more commonly available phosphoric acid instead. It is less aggressive than either nitric or sulfuric at the same concentration.

Acid scarified 16 SM seeds with 85% phosphoric acid for 3 minutes. Rinsed 3x with tap water. Sown onto filter paper in a 90mm petri dish. Placed in the incubator at 30°C.

Notes

Bibliography

1. Lercari, B. and Sponga, F., Effect of Chemical Treatments on the Photocontrol of Germination in «Atropa Belladonna» L. Seeds, Rivista di ortoflorofrutticoltura italiana, vol. 69, no. 2, pp. 115--122, 1985. url: https://www.jstor.org/stable/42878729. Abstract
   The effects of many chemicals (fusicoccin, GA₃, GA4/7, abscissic acid (ABA), cycloheximide and actinomycin D) on the light-mediated germination of the seeds of A. belladonna were studied. Fusicoccin cannot induce seed germination in the dark, but it enhances the percentage germination either in the dark or under continuous red light. It appears that the two factors red light and GA act independently. ABA and cycloheximide inhibit completely the light mediated germination, while the effect of actinomycin D is very low. It is suggested that phytochrome-mediated seed germination in A. belladonna requires cytoplasmic protein-synthesis, while there are no indications for a requirement in RNA synthesis. Our results exclude the likelihood that phytochrome controls germinations of Atropa belladonna seeds through the activation of a H⁺/K⁺ exchange mechanism, fusicoccin sensitive, at the membrane level. Si è studiato l'effetto di vari composti chimici (fusicoccina, GA₃, GA4/7, acido abscissico (ABA), cicloesimide e actinomicina D) sulla germinazione dei semi di A. belladonna indotta dalla luce. La fusicoccina non è in grado di indurre la germinazione al buio, ma la aumenta alla luce. GA₃ e GA4/7 sono in grado di stimolare la germinazione sia alla luce che al buio. I due fattori luce e gibberelline sembrano agire indipendentemente l'uno dall'altro nel controllo della germinazione di A. belladonna. L'acido abscissico e la cicloesimide inibiscono completamente la germinazione indotta dalla luce, mentre l'effetto della actinomicina D è scarso o nullo. I risultati suggeriscono che in A. belladonna la germinazione mediata dal fitocromo richiede sintesi proteica citoplasmatica e sembra non richiedere l'attivazione a livello di membrana di un meccanismo di scambio H⁺/K⁺ fusicoccina sensibile.
2. {Asha Rani N.S} and {Prasad M.P}, In-Vitro Studies on the Germination of Atropa Belladonna Seeds under Different Conditions, International Journal of Science and Research, vol. 3, no. 10, 2012. url: https://www.ijsr.net/archive/v3i10/T0NUMTQxODQ=.pdf. Abstract
   Atropa belladonna contains tropane alkaloids and a raw material for pharmaceutics industry. Large scale cultivation of Belladonna is carried both by bulb and seeds. Seeds are usually sown during the first half of March and it take almost 3 months for germination and hence farmers prefer bulbs of belladonna for cultivation. Prolong germination of seed is due to seed Dormancy. Dormancy is a mechanism to prevent germination during unsuitable ecological conditions, when the probability of seedling survival is low. The present investigation was carried out to improve germination percentage of Atropa belladonna seeds by different Scarification methods. Mechanical and Chemical Scarifications were carried on seeds of belladonna. Both mechanical and chemical methods significantly stimulated seed germination in varying percentage. Seeds were germinated on full strength M S media with or without GA3 under invitro condition. Maximum germination was obtained by Acid treatments and also by boiling water treatments. Hard impermeable Testa of the seeds was successfully broken by above Scarification treatment and even insufficient germinating hormones were also supplied for good germination percentage in present investigation.
3. Smolenski, Stanislaus J. and Crane, Frank A. and Voigt, Ralph F., Effects of the Ratio of Calcium to Potassium in the Nutrient Medium on the Growth and Alkaloid Production of Atropa Belladonna, Journal of Pharmaceutical Sciences, vol. 56, no. 5, pp. 599--602, May 1967. doi: 10.1002/jps.2600560511. Abstract
   The increase of calcium/potassium ratios in nutrient solutions reduces growth of belladonna plants. This is evident in the reduced elongation of all stems, particularly the sympodial flowering branches, and in the fresh and dry weights of all plant parts. There appears to be a concurrent increase in the proportion of leaf to total plant at the expense of stem and root. The increase in calcium/potassium ratio results in higher yields of total nitrogen and alkaloids.
4. Schermeister, L. J. and Crane, F. A. and Voigt, R. F., Growth and Differentiation of Atropa Belladonna L. as Affected by Different Sources of Nitrogen, Journal of the American Pharmaceutical Association, vol. 49, no. 11, pp. 694--697, 1960. doi: 10.1002/jps.3030491104. Abstract
   Growth and morphology were compared on plants furnished six levels of nitrate and three of ammonia in water culture. The production of dry weight with ammonia was significantly greater than with nitrate. Ratios of shoot/root and leaf/stem indicated that use of the nitrogen was very different at low vs. high levels, particularly with ammonia nitrogen.
5. Genova, Elena and Komitska, Gergana and Beeva, Yundina, Study on the Germination of Atropa Bella-Donna L. Seeds, Bulgarian Journal of Plant Physiology, vol. 23, no. 1-2, pp. 61--66, 1997. url: http://www.bio21.bas.bg/ipp/gapbfiles/v-23/97_1-2_61-66.pdf. Abstract
   The germination of Atropa bella-donna L. seeds, a medicinal species with restrictive mode of use, was studied. It was established that variable temperature (6 h at 30 °C and 18 h at 15 °C) significantly stimulates seed germination – 82.5\%. A maximum germination was obtained by treatment with gibberellic acid (GA3) 1mg/l H2O – 89.5\%
6. Kutlymuratova, G. A., Ecological Features and Agrotechnology of Growing Atropa Belladonna l. and Echinops Ritro L in the Conditions of Karakalpakstan, Journal of Pharmaceutical Negative Results, pp. 2400--2404, November 2022. doi: 10.47750/pnr.2022.13.S08.297. Abstract
   The article deals with the study of ecological features and agrotechnology of growing Atropa belladonna L. and Echinops ritro L in the conditions of the Republic of Karakalpakstan. The adaptive adaptability is the highest in Atropa belladonna L., the plant has a high combined resistance to local climatic conditions, massively blooms and bears fruit, actively self-propagates vegetatively.
7. Hartmann, T. and Witte, L. and Oprach, F. and Toppel, G., Reinvestigation of the Alkaloid Composition of Atropa Belladonna Plants, Root Cultures, and Cell Suspension Cultures, Planta Medica, vol. 52, no. 5, pp. 390--395, October 1986. doi: 10.1055/s-2007-969194. Abstract
   Thieme E-Books \& E-Journals
8. Brewer, W. R. and Hiner, L. David, Cultivation Studies of the Solanaceous Drugs. II. The Effect of Nutritional and Soil Reaction Fertilizers on the Production Yields and Total Alkaloidal Content of Atropa Belladonna and Hyoscyamus Niger, Journal of the American Pharmaceutical Association, vol. 39, no. 10, pp. 586--591, 1950. doi: 10.1002/jps.3030391015. Abstract
   In order to clarify some of the contradiction and confusion existing in the literature concerning the value of adjusting pH, and nitrogen, phosphorus, and potassium levels in solanaceous plants and their alkaloid production, a study was begun with belladonna and hyoscyamus plants growing in controlled variations of these elements and their concentrations. The results of these initial studies were subjected to statistical interpretation to determine their significance.
9. Rita, Paul and Datta, Animesh, An Updated Overview on Atropa Belladonna L, International Research Journal of Pharmacy, vol. 2, pp. 11--17, November 2011. Abstract
   Atropa belladonna L. (Family: Solanaceae; commonly known as belladonna, deadly nightshade, devil’s berries amongst others), a perennial herb (2n=72) is native of Europe, North Africa and Western Asia, possesses a long tradition as one of the classic poisons of antiquity. The species is also the source of atropine alkaloid (dl-hyoscyamine) and is important in the study of autonomic pharmacology. Considering the therapeutic uses of A. belladonna as well as its significant toxic effects (due to tropane alkaloids including scopolamine and hyoscyamine), an overview on all necessary aspects is documented to provide information for further exploration of the species for human benefits.
10. Baricevic, Dea and Umek, Andrej and Kreft, Samo and Maticic, Branivoj and Zupancic, Alenka, Effect of Water Stress and Nitrogen Fertilization on the Content of Hyoscyamine and Scopolamine in the Roots of Deadly Nightshade (Atropa Belladonna), Environmental and Experimental Botany, vol. 42, no. 1, pp. 17--24, August 1999. doi: 10.1016/S0098-8472(99)00014-3. Abstract
    The study intended to elaborate the optimal environmental conditions of water supply and nitrogen fertilization for maximum content of hyoscyamine (\% dw) and scopolamine (\% dw). Plants grown from seeds of Slovene autochthonous population of deadly nightshade (Atropa belladonna), were treated with different water regimes (35–95\% depletion of available soil water) together with enhanced nitrogen supply (0.37–1.60 g/pot N) in a greenhouse experiment. Dry plant extracts from 32-week old roots were analysed with capillary electrophoresis (CE) for the presence of tropane alkaloids (hyosciamyne, scopolamine). The results of the plant treatment responses showed that the maximal yield of tropane alkaloids (hyoscyamine: 54 mg/plant; scopolamine: 7 mg/plant) was achieved in plants grown under an optimal irrigation regime (35\% depletion of available soil water) accompanied with total nitrogen supply of 0.37 g/pot. By contrast, the maximal content of alkaloids was achieved with 95\% depletion of available soil water and a nitrogen supply of 1.60 g/pot.
11. Elzenga, G. and Smeets, L. and De Bruyn, J. W., Influence of the Temperature on Growth and Alkaloid Content of First-Year Atropa Belladonna L., Euphytica, vol. 5, no. 3, pp. 276--280, October 1956. doi: 10.1007/BF00038849. Abstract
    In breeding Atropa belladonna for high alkaloid content it is of importance to know whether the alkaloid content is affected by the environment. Therefore some experiments on the influence of temperature were carried out in the phytotron of our Institute. As the results were in agreement with each other only one experiment is discussed in which the influence of three constant temperatures, viz. 20°, 23° and 26°C, on growth rate, dry matter, and alkaloid content of yellow-flowered Atropa belladonna L. was studied.
12. Sporer, F. and Sauerwein, M. and Wink, M., Diurnal and Developmental Variation of Alkaloid Accumulation in Atropa Belladonna, Acta Horticulturae, no. 331, pp. 381--386, September 1993. doi: 10.17660/ActaHortic.1993.331.53. Abstract
    The production and distribution of tropane alkaloids in organs of wild Atropa belladonna plants was studied by HPLC. Two different growth stages were examined in June and July and in addition berries and seeds in September 1991. A seasonal variation in total alkaloid content and alkaloid patterns of these plant parts was observed. Hyoscyamine was the main product throughout. A diurnal variation of tropane alkaloid content could be determined. Two peaks were significant: maximal alkaloid yields were detected at early night and at early morning. In maturing seeds, the alkaloid content was highest in the afternoon.
13. Cromwell, B. T., Studies on the Synthesis of Hyoscyamine in Atropa Belladonna L. and Datura Stramonium L, Biochemical Journal, vol. 37, no. 6, pp. 717--722, 1943. url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1258005/.
14. {Kohnen-Johannsen}, Kathrin Laura and Kayser, Oliver, Tropane Alkaloids: Chemistry, Pharmacology, Biosynthesis and Production, Molecules, vol. 24, no. 4, pp. 796, February 2019. doi: 10.3390/molecules24040796. Abstract
    Tropane alkaloids (TA) are valuable secondary plant metabolites which are mostly found in high concentrations in the Solanaceae and Erythroxylaceae families. The TAs, which are characterized by their unique bicyclic tropane ring system, can be divided into three major groups: hyoscyamine and scopolamine, cocaine and calystegines. Although all TAs have the same basic structure, they differ immensely in their biological, chemical and pharmacological properties. Scopolamine, also known as hyoscine, has the largest legitimate market as a pharmacological agent due to its treatment of nausea, vomiting, motion sickness, as well as smooth muscle spasms while cocaine is the 2nd most frequently consumed illicit drug globally. This review provides a comprehensive overview of TAs, highlighting their structural diversity, use in pharmaceutical therapy from both historical and modern perspectives, natural biosynthesis in planta and emerging production possibilities using tissue culture and microbial biosynthesis of these compounds.
15. Huang, Jian-Ping and Wang, Yong-Jiang and Tian, Tian and Wang, Li and Yan, Yijun and Huang, Sheng-Xiong, Tropane Alkaloid Biosynthesis: A Centennial Review, Natural Product Reports, vol. 38, no. 9, pp. 1634--1658, September 2021. doi: 10.1039/D0NP00076K. Abstract
    Covering: 1917 to 2020 Tropane alkaloids (TAs) are a remarkable class of plant secondary metabolites, which are characterized by an 8-azabicyclo[3.2.1]octane (nortropane) ring. Members of this class, such as hyoscyamine, scopolamine, and cocaine, are well known for their long history as poisons, hallucinogens, and anaesthetic agents. Since the structure of the tropane ring system was first elucidated in 1901, organic chemists and biochemists have been interested in how these mysterious tropane alkaloids are assembled in vitro and in vivo. However, it was only in 2020 that the complete biosynthetic route of hyoscyamine and scopolamine was clarified, and their de novo production in yeast was also achieved. The aim of this review is to present the innovative ideas and results in exploring the story of tropane alkaloid biosynthesis in plants from 1917 to 2020. This review also highlights that Robinson's classic synthesis of tropinone, which is one hundred years old, is biomimetic, and underscores the importance of total synthesis in the study of natural product biosynthesis.
16. Suzuki, K.-i. and Yamada, Y. and Hashimoto, T., Expression of Atropa Belladonna Putrescine N-Methyltransferase Gene in Root Pericycle, Plant and Cell Physiology, vol. 40, no. 3, pp. 289--297, January 1999. doi: 10.1093/oxfordjournals.pcp.a029540.
17. Choudhary, D., Causes of Poor and Erratic Germination in Atropa Belladonna, Planta Medica, vol. 27, no. 01, pp. 18--22, February 1975. doi: 10.1055/s-0028-1097754. Abstract
    An effort has been made to arzalyse the probable causes of a low germination o f Atropa species. From the results achieved it is expected that the very hard, thick, woody and waxy nature of testa along with an internal membrane enclosing the embryo and the presence .of seeds containing only a rudimentary embryo may be the va/ious factors responsible for poor and erratic gernlination of Atropa species.